This application requests funds to purchase a multi-use fluorescence microscope to be shared by three NIH R01 supported Principal Investigators and one NIH K99 supported investigator in the Department of Biochemistry at Stanford University School of Medicine. The laboratories of Jim Spudich, Suzanne Pfeffer, Aaron Straight and Rajat Rohatgi share a common interest in microscopy based dynamic analysis of cellular systems and mechanistic dissection of those systems through biochemical analysis. Central to the research in our laboratories is the use of fluorescence microscopy to study cellular protein and organelle dynamics. In order to analyze complex cell biological processes, our laboratories combine optical, chemical, and genetic perturbation of cells with high-resolution immunofluorescence imaging and live cell timelapse imaging. To understand how individual cellular components participate in cellular functions we apply microscopy-based analysis to reconstituted biochemical systems and cell extract based reactions that recapitulate cellular processes. Specifically, we use methods ranging from single molecule analysis of motor protein movement on cytoskeletal polymers by optical trapping and TIRF microscopy to epifluorescence imaging of chromosome segregation and vesicle trafficking in cell extracts. We are requesting funds to purchase a shared microscope capable of live cell time-lapse fluorescence and brightfield microscopy, TIRF microscopy, patterned fluorescence photobleaching and photoactivation and iterative deconvolution. These combined modalities will significantly enhance the NIH supported research in our laboratories by providing a shared resource to address our microscopy needs.